How to distinguish between 16S chloroplast reads and real ones?

Light92 · 11-19-2019, 01:20 AM

Hello,

In an old experiment dating back to 2014, some 16S libraries from plant samples were prepared, with the aim to identify the microbial community living/contaminating the surface of the fruits.

The primer choice was the couple 27F and 533R.

Sadly, once analyzed with QIIME2 and clustered, out of 12 samples, 10 had chloroplast DNA contaminations percentages up to 99% (80-99%), making them completely useless.

I'd like to run the experiment again, with Illumina technology, addressing the problem of the primers not being specific enough to distinguish between plastid and bacteria DNA.

At the moment, the only scientific paper directly addressing the issue I was able to find was this one, but it's quite an old one.

Any suggestions here?
Thanks in advance.

seanw · 11-26-2019, 06:11 AM

I too am having the issue when looking for endophytes in leaf and stem tissue. Qiagen DNeasy as well as Qiagen Kingfisher soil kit extractions then using V4 region as well as V5/6 are not working well for me on our MiSeq. What are others doing to reduce chloroplast and yet have a good and reliable region to survey?

ATϟGC · 11-26-2019, 12:14 PM

Have you tried or considered using a blocking oligo (such as one with a C3 spacer) designed to reduce amplification of the "host" plant tissue? A quick look at an alignment of nucleotide data might tell you whether there are any good candidate blocking oligo sites.

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11-19-2019, 01:20 AM	#1
Light92 Junior Member Location: Italy Join Date: Sep 2019 Posts: 5	How to distinguish between 16S chloroplast reads and real ones? Hello, In an old experiment dating back to 2014, some 16S libraries from plant samples were prepared, with the aim to identify the microbial community living/contaminating the surface of the fruits. The primer choice was the couple 27F and 533R. Sadly, once analyzed with QIIME2 and clustered, out of 12 samples, 10 had chloroplast DNA contaminations percentages up to 99% (80-99%), making them completely useless. I'd like to run the experiment again, with Illumina technology, addressing the problem of the primers not being specific enough to distinguish between plastid and bacteria DNA. At the moment, the only scientific paper directly addressing the issue I was able to find was this one, but it's quite an old one. Any suggestions here? Thanks in advance.

11-26-2019, 06:11 AM	#2
seanw Junior Member Location: New Brunswick, Canada Join Date: Nov 2014 Posts: 3	I too am having the issue when looking for endophytes in leaf and stem tissue. Qiagen DNeasy as well as Qiagen Kingfisher soil kit extractions then using V4 region as well as V5/6 are not working well for me on our MiSeq. What are others doing to reduce chloroplast and yet have a good and reliable region to survey?

11-26-2019, 12:14 PM	#3
ATϟGC Member Location: Canada Join Date: Jun 2013 Posts: 51	Have you tried or considered using a blocking oligo (such as one with a C3 spacer) designed to reduce amplification of the "host" plant tissue? A quick look at an alignment of nucleotide data might tell you whether there are any good candidate blocking oligo sites.